Species-specific Primers Research Articles

Mycotoxigenic Fusarium species and zearalenone concentration in commercial maize kernels in northern Ghana.

The fungal genus Fusarium contains many toxigenic pathogens of maize with associated yield losses, reduction of grain quality, and accumulation of mycotoxins in harvested grains. To determine zearalenone (ZEN) concentration and identify the various Fusarium species in commercial maize grains, a survey of 75 maize samples, collected from 11 market centers in the five regions in northern Ghana was identified based on morphological characteristics, sequence analysis of the internal transcribed spacer region, and polymerase chain reaction using species-specific primers. ZEN levels were determined using HPLC. ZEN contamination was recorded in 33.3% of the maize samples, with concentrations ranging from 0.61 to 3.05µg/kg. Based on VERT1/2 and TEF 1-α sequencing, F. verticillioides was the most prevalent species in the studied samples: 40.35% from the Upper East Region, 28.07% from the North East Region, 19.30% from the Upper West Region, 10.53% from the Savannah Region, and 1.75% for the Northern Region. Other fungal species found were F. equiseti and F. solani. A higher number of the Fusarium isolates were found in white maize (609 isolates from 27 samples) compared to yellow maize (225 isolates from 23 samples).

The employment of species-specific primer in combination with real-time polymerase chain reaction for analysis of pork in meatball products

In line with the implementation of Indonesian Act No. 33 year 2014 on Halal Products Assurance, the detection of a non-halal component in food products is critical to confirm the authenticity of halal products. The aim of this study was to employ real-time polymerase chain reaction (real-time PCR) using species-specific primer targeting D-loop mitochondria for analysis of pork DNA in meatballs. The real-time PCR technique was evaluated for the primer specificity, amplification efficiency, and sensitivity as determined by evaluating the limit of detection (LoD). The designed primer was specific to pork DNA, with an amplification efficiency of 82.7%. The developed method could detect DNA in meatballs as low as 10 ng DNA. The reproducibility of real-time PCR was acceptable, as indicated by the low relative standard deviation (RSD) of 1.81%. The developed method was successfully applied to analyze pork in meatballs available in the commercial market. Real-time PCR using species-specific primer targeting D-loop mitochondria offered a reliable method for the detection of pork DNA in meatball products and can be proposed as a standard method for supporting the authenticity of halal products.

Morphological and Molecular Identification of Root-knot Nematode, Meloidogyne incognita Infecting Pomegranate (Punica granatum L.) in Jodhpur, Rajasthan, India

Root-knot nematodes (Meloidogyne spp.) pose a major threat to pomegranate (Punica granatum L.) cultivation in India, leading to significant yield losses. In this study, galled roots of pomegranate were collected from orchards in Jodhpur, Rajasthan and nematode infestation was confirmed through root staining. The perineal pattern of females displayed the typical characteristics of the Meloidogyne incognita. To further confirm the species, DNA was extracted and a polymerase chain reaction (PCR) assay using species-specific SCAR (sequence-characterized amplified region) primers was conducted. The M. incognita- species specific primers, MincF1/MincR1, yielded the expected 150 bp product, verifying the presence of M. incognita. This study marks the first molecular confirmation of M. incognita infecting pomegranate orchards in this region, demonstrating the efficacy of SCAR markers in complementing traditional morphological identification as well.

Identification of Geotrichum citri-aurantii and G. candidum in citrus packinghouses in California, their sensitivity to cyproconazole, and incomplete cross-resistance to propiconazole.

High levels of sour rot on propiconazole-treated lemon fruit that were stored for extended times in some California packinghouses in 2020 and 2021 initiated surveys on fungicide sensitivity of the causal pathogen. In isolations from diseased fruit in 2020 to 2023, 157 isolates of Geotrichum spp. were obtained. Using species-specific primers, 143 were determined to be G. citri-aurantii and 15 were G. candidum. Isolates of G. citri-aurantii were either sensitive (EC50 0.06 to 0.34 μg/ml), moderately resistant (EC50 1.20 to 2.34 μg/ml), or highly resistant (EC50 >17.68 μg/ml) to propiconazole. There was incomplete cross resistance to cyproconazole, another demethylation inhibitor fungicide pending postharvest registration on citrus in the United States: isolates sensitive to propiconazole were sensitive, isolates moderately resistant to propiconazole were sensitive, and isolates highly resistant were moderately resistant to cyproconazole (EC50 0.11 to 0.63 μg/ml, 0.19 to 0.73 μg/ml, and 2.66 to 6.79 μg/ml, respectively). All except one isolate of G. candidum were highly resistant to both fungicides (EC50 >9.55). Isolates of both species were all considered sensitive to natamycin (EC50 1.18 to 5.01 μg/ml). In lemon fruit inoculations with G. candidum, the incidence of typical sour rot increased from 4.7% to 68.2% when inoculum was amended 2 µg/ml or 100 µg/ml cycloheximide, respectively, a compound known to suppress host defenses. In co-inoculations with 1:1 mixtures of the two Geotrichum spp., G. candidum was only recovered from the centers of decay lesions, whereas G. citri-aurantii was also obtained from the advancing margins. We conclude that G. candidum is a secondary pathogen of lemons, and its presence was favored by extended late-season storage of senescent fruit with reduced defense mechanisms.

Potentially Pathogenic Vibrio spp. in Algal Wrack Accumulations on Baltic Sea Sandy Beaches

The Vibrio bacteria known to cause infections to humans and wildlife have been largely overlooked in coastal environments affected by beach wrack accumulations from seaweed or seagrasses. This study presents findings on the presence and distribution of potentially pathogenic Vibrio species on coastal beaches that are used for recreation and are affected by red-algae-dominated wrack. Using species-specific primers and 16S rRNA gene amplicon sequencing, we identified V. vulnificus, V. cholerae (non-toxigenic), and V. alginolyticus, along with 14 operational taxonomic units (OTUs) belonging to the Vibrio genus in such an environment. V. vulnificus and V. cholerae were most frequently found in water at wrack accumulation sites and within the wrack itself compared to sites without wrack. Several OTUs were exclusive to wrack accumulation sites. For the abundance and presence of V. vulnificus and the presence of V. cholerae, the most important factors in the water were the proportion of V. fucoides in the wrack, chl-a, and CDOM. Specific Vibrio OTUs correlated with salinity, water temperature, cryptophyte, and blue-green algae concentrations. To better understand the role of wrack accumulations in Vibrio abundance and community composition, future research should include different degradation stages of wrack, evaluate the link with nutrient release, and investigate microbial food-web interactions within such ecosystems, focusing on potentially pathogenic Vibrio species that could be harmful both for humans and wildlife.

Use of Environmental DNA to Evaluate the Spatial Distribution of False Kelpfish (Sebastiscus marmoratus) in Nearshore Areas of Gouqi Island

This study aims to explore the spatial distribution of false kelpfish (Sebastiscus marmoratus) in the mussel farming area, artificial reef areas of Gouqi Island (Shengsi, China), and natural areas using eDNA detection methods. Surface and bottom water samples were collected at 12 stations in November 2022 and April 2023, totaling 52 samples. We used species-specific primers and probes for quantitative PCR (qPCR) detection of Sebastiscus marmoratus eDNA. The eDNA concentrations differed seasonally (p < 0.05) and did not differ (p > 0.05) among the three sampling areas and two water layers. The greatest eDNA concentrations occurred in the surface layer during the spring. Higher concentrations of Sebastiscus marmoratus eDNA were also found in the mussel aquaculture area. Temperature exhibited a significant positive correlation with Sebastiscus marmoratus eDNA concentration (p < 0.05). Additionally, we developed linear equations predicting the relationship between environmental factors and environmental factors, providing a reference for future fishery resource surveys.

Early Molecular Detection of Invasive Alien Plants in Urban and Peri-Urban Areas

Invasive alien plants represent one of the five major threats to biodiversity and the disruption of ecosystems. They are introduced through various routes, starting with commercial trade. Preventing their introduction is essential to avoid the spread of new invasive plants. In this paper, we propose a new early warning DNA barcoding tool for invasive plant detection. Eight invasive alien species of European Union concern (i.e., Ludwigia grandiflora, Elodea nuttallii, Myriophyllum aquaticum, Pontederia crassipes, Ailanthus altissima, Heracleum mantegazzianum, Impatiens glandulifera, Pueraria montana) were selected and analysed. A unique DNA marker for each species was identified and amplified using species-specific primers capable of identifying the presence of alien species. To verify whether the approach could detect the presence of alien plants in urban areas from lawn clippings, mixes with typical urban spontaneous plants and invasive species were tested. In all mixes, only the invasive species was identified. This rapid detection capability will enable environmental operators to intervene promptly to contain the spread of invasive plants before they can cause significant damage to the local ecosystem. This tool could have a significant impact on the protection of local biodiversity and the integrity of urban habitats.

First Report of Fire Blight Caused by Erwinia amylovora on Pear in Saudi Arabia.

In the spring of 2020 and 2021, pear trees (Pyrus communis cv. Williams) in orchards (27°46'36.0"N 42°29'44.7"E, 30°00'00.2"N 40°15'11.8"E, and 28°44'52.9"N 36°18'47.8" E) in Hail, Al Jouf, and Tabuk regions exhibited fire blight symptoms. After removing bark, the affected trees showed shoot blight, brown-blighted shoot tips and blossom blight, dead flowers on the stems, and reddish-colored cankers. The disease incidence varied from 10% to 25%. Pathogen was isolated from 21 symptomatic samples including fruits, flowers, and shoots. Bacteria were isolated from washed tissues on King's B (KB) and semi-selective CCT media (Ishimaru and Klos, 1984). After 48 hours, colonies resembling Erwinia amylovora on KB media were 1.5-2 mm in diameter, white, circular, slightly convex, with a smooth surface, and exhibited no fluorescence under ultraviolet light. Colonies on CCT after 72 h were 3-4 mm in diameter, mucoid with shiny surfaces, semitransparent, speckled with craters, and slightly violet. All isolates were purified by subculturing on KB. All isolates were Gram-negative and rod-shaped, fermentative of glucose, positive for catalase and negative for oxidase, and potato rot. They induced a hypersensitive reaction when infiltrated in tobacco leaves (cv. Xanthi). Based on morphology and biochemical tests (EPPO, 2004), the strains were identified as Erwinia sp. Twenty-six strains from Saudi Arabia (SA) and the reference strain (NCPPB 683T) hydrolyzed gelatin and formed white, highly mucous colonies on the levan medium. These strains could not reduce nitrate to nitrite and tested negative for urease and indole production. All the isolates and the reference strain were confirmed to be E. amylovora based on a PCR 0.9-kb DNA fragment amplification with a species-specific primer set, A/B targets pEA29 (Bereswill et al. 1992). 16S-rDNA fragments from Saudi isolates were amplified with 27F and 1492R primers (Lane, 1991). Purified amplicons from PCR were sequenced (OR717505 and OR743536-OR743560), and a BLAST search of the GenBank database revealed 100% (927/927) homology with E. amylovora strain CP066796.1. The housekeeping gene rpoB was PCR amplified with primers CM7-F and CM31b-R (Rezzonico et al. 2012), and the products were sequenced (PP465516-PP465541). BLAST analysis showed 100% (944/944 nt) and 96.19% (908/944 nt) identities with the sequences of E. amylovora ATCC 15580 CP066796.1 and E. pyrifoliae CP201486 CP103445.1, respectively. To fulfill Koch's postulates, SA strains were inoculated on five healthy 3-month-old clones of apple (Malus domestica cv. Gala) per strain with a 10-μl bacterial suspension containing 107 colony-forming units per milliliter by injecting directly in the veins of the upper second leaf plus 5 healthy plants injected with sterile distilled water as control. Plants were incubated at 28°C for six days under a 12-hour light regime. Observed symptoms were similar to the ones observed in the field. The experiment was replicated twice. Bacterial colonies on CCT media were re-isolated from the inoculated apple rootstocks and confirmed by the A/B primer set. To our knowledge, this is the first peer-reviewed report of E. amylovora in SA since the fire blight-like symptoms were observed in SA in 2013 (Alhudaib, 2013). Further research will identify new host plants for the fire blight pathogen within SA which is important due to the importation of pome fruit seedlings (quince, apple, and pear) from neighboring Jordan where E. aylovora was reported (Tehabsim et al. 1992).

Development of molecular diagnostic protocols for simultaneous identification of common bed bugs (Cimex lectularius) and tropical bed bugs (Cimex hemipterus)

BackgroundThe resurgence of two bed bug species, the common bed bug (Cimex lectularius Linnaeus, 1758) and tropical bed bug (Cimex hemipterus Fabricius, 1803), in the same geographical regions has been frequently reported recently. Consequently, the rapid identification of these species is crucial for implementing targeted capture traps and tailored pyrethroid resistance diagnosis, due to differences in genetic and physiological traits.MethodsTo develop molecular diagnostic methods, distinct protocols were established for multiplex PCR and loop-mediated isothermal amplification (LAMP) using species-specific primers based on species-specific segments of internal transcribed spacer 2 sequences. These methods were optimized for rapid and accurate identification of the two bed bug species.ResultsBoth multiplex PCR and LAMP protocols were effective in simultaneously identifying the two bed bug species, even when utilizing DNA released from dead specimens. Notably, the straightforward procedure and minimal time commitment of LAMP suggest its potential for rapid and accurate diagnosis of bed bugs in the field. The diagnostic accuracy of these methods was validated through a blind test.ConclusionsThe multiplex PCR and LAMP protocols lay the foundation for rapid and accurate field identification of bed bug species, enabling the use of appropriate traps and the detection of species-specific pyrethroid resistance mutations. This approach ensures effective management tailored to the unique characteristics of each bed bug species.Graphical

Detection of human strongyloidiasis among patients with a high risk of complications attending selected tertiary care hospitals in Colombo, Sri Lanka using molecular and serological diagnostic tools

BackgroundStrongyloidiasis a neglected tropical disease is known to cause severe disease among immunosuppressed and has not been studied extensively in Sri Lanka. Parasitological diagnostic approaches based on faecal microscopy and culture often fail to detect low-intensity infections. This study investigates the presence of strongyloidiasis among selected immunocompromised individuals using parasitological, molecular and serological techniques.MethodsAdult patients with immunocompromising conditions admitted to three tertiary care hospitals in Sri Lanka were recruited. A faecal sample and 2 ml of venous blood were collected. The faecal samples were subjected to direct faecal smear and cultures (agar plate, charcoal and Harada-Mori) and polymerase chain reaction (PCR) using species specific primers designed for Strongyloides stercoralis. The presence of Strongyloides IgG antibodies was tested in the collected serum samples using DRG Strongyloides IgG enzyme-linked immunosorbent assay (ELISA) kits. The PCR products of the positive samples were sequenced using Sanger sequencing method.ResultsA total of 260 patients were recruited to this study, out of which 160 provided faecal samples and 122 provided blood samples. Out of the 160 faecal samples, none were positive for strongyloidiasis by direct smear, charcoal and Harada-Mori cultures. Only one sample (0.6%) was positive by agar plate culture. Out of the 123 samples subjected to PCR, 14 (11.4%), including the culture positive patient, were positive for S. stercoralis. Sequencing results of the PCR products indicated 100% similarity to S. stercoralis. Out of the 122 serum samples subjected to ELISA, 20 (16.4%), including the culture positive patient, were positive for Strongyloides IgG antibodies. However, sociodemographic, exposure factors, clinical features were not significantly associated with the presence of strongyloidiasis infection.ConclusionsStrongyloidiasis is present among the immunocompromised population in Sri Lanka, even in the absence of a significant relationship with associated factors. It is advisable to screen such patients with highly sensitive tests such as PCR for early diagnosis and treatment.Graphical

Full-genome characterization of Turkish Rose Yellow Vein Virus (RYVV) Isolates

Rose yellow vein virus (RYVV) is a viral agent that has been identified in roses in recent years and is known to exist in a very limited area in the world. However, in studies conducted in our country, the presence of the agent has been detected in different regions and provinces. Since direct chemical control of viral diseases is not possible as in other biotic diseases, molecular characterization of the agent forms the basis for possible resistance or gene silencing studies to be conducted in the coming years. Thus, 44 RYVV isolates previously identified in our country were used for full genome studies. In full genome studies, overlapping PCR method was used with 7 different species-specific primer pairs. The full genomes of 3 of these isolates were obtained. The full genomes of the other isolates could not be obtained. It was determined that the genome size of the 3 RYVV isolates whose full genomes were obtained was 9314 nucleotides. It was determined that the only RYVV isolate found in the GenBank and the Türkiye isolates also showed a high level of nucleotide sequence similarity. However, the fact that the entire genome of the other isolates could not be obtained and the ones obtained were amplified with primer pairs designed according to the single isolate in the GenBank database may be due to the presence of different strains of RYVV isolates. For this reason, it is thought that next-generation sequencing studies should be prioritized to obtain complete genome information in future studies.

Development of species-specific multiplex PCR for Leishmania identification

Leishmaniasis is a diverse group of clinical diseases caused by protozoan parasites of the Leishmania genus. Species-specific identification of Leishmania spp. is challenging due to the high number of different pathogenic species that sometimes co-circulate in the same foci, hampering efforts to effectively control the disease. Multiplex PCR is an attractive alternative for rapid differentiation of Leishmania species with high sensitivity and specificity. We aimed to generate a panel of primers optimized for a multiplex PCR assay capable of identifying different Leishmania species in a single reaction. Species-specific primers were designed based on genomic data using the TipMT tooL. Potential non-specific amplifications of other trypanosomatids as well as human, dog, and sandfly hosts were first evaluated in silico using the Primer-Blast tooL. Species-specific primers for Leishmania amazonensis, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania mexicana and for the Leishmania guyanensis complex were tested in vitro. The primers have a limit of detection ranging from 1 to 0.01 ng of parasite gDNA using the same annealing temperature of 66 °C. The primers were specific for their targets when tested against 13 species of Leishmania, six trypanosomatids, and Babesia sp., and to detect the target species in a prepared pool with gDNA of six pathogenic Leishmania species. The designed primers were optimized for multiplex PCR, enabling species-specific identification of all five Leishmania species and one species complex. This new primer set could allow for efficient, fast, and reliable identification of Leishmania parasites.

Rapid identification of Radix Achyranthis Bidentatae and Radix Cyathulae ingredients using Proofman-LMTIA method

Radix Achyranthis Bidentatae (RAB) and Radix Cyathulae (RC) share the same name “Niu-Xi”, which are two types of well-known herbal medicines in Asian countries due to their long history and extensive pharmacological activities. However, the misuse of counterfeits and confused species of RAB or RC due to the increased demands might lead to improper efficacies and serious health risks to consumers. To address the challenge, this study devises a rapid and accurate method named Proofman-LMTIA for the identification of RAB and RC. A set of species-specific primers were selected to target internal transcription spacer (ITS) gene sequences of RAB and RC. The temperatures of the Proofman-LMTIA systems were optimized, and the specificities, sensitivities and detection limits were investigated. The detection of RAB and RC ingredients based on the Proofman-LMTIA method was finished in less than 20 min. The sensitivities of RAB and RC DNA templates were 50 pg/µL and 10 pg/µL, respectively. The detection limits of RAB and RC DNA samples extracted from artificially adulterated formula granules were 0.1 %. To evaluate the applicability and effectiveness of the Proofman-LMTIA systems, 16 formula granules were collected and analyzed for RAB or RC authentication, among these, 14 out of 16 samples tested positive for RAB or RC components. Overall, the Proofman-LMTIA technique could be a reliable and suitable method for the accurate and rapid identification of RAB and RC ingredients in commercial samples. The popularization of the LMTIA technique will protect the rights of consumers and maintain the stability of the medicine market.

Molecular Identification of Normal and Adulterated Meat Samples of Local Markets and Restaurants in Kerbala Province

Background: Species specification of the meat is a highly important field of quality control management in meat consumption and industry. Meat forgery with undeclared sources has become a crucial trouble in many countries as well as in Iraq. It means that raw consumed meat or meat derivatives may include different meat genus; so, the components of meat are not regular like the known marker. The diet has sever effect on public health, religious factor, food safety principles, fair-trades and consumers desire. Aim of the study: The current study was aimed to analysis the species of adulterated meat using molecular technique that targets Mitochondrial Cytochrome cyt b gene by Multiplex PCR for detection of meat species and authentication that provide many forensic and judicial applications for several populations throughout the world. Materials and Methods: The following seventy (70) typical meat and meat derivatives were gathered from neighborhood super markets and eateries in the province of Kerbala: 25 cow, 10 buffalo, 25 sheep, and 10 goats. For meat samples, DNA extraction was done. Next, using species-specific primers of the Mitochondrial Cytochrome cyt b gene for cattle, sheep, goats, and buffalo, the collected DNA was amplified using the Multiplex PCR method. Additionally, the presence of Staphylococcus aureus, one of the microorganisms most commonly linked to food illness, was typically examined in all of the meat samples. Results: Multiplex PCR technique resulting in species-specific products of the amplicon sizes for the DNA prepared samples with the lengths 472bp., 124bp., 585bp., and 330bp. for the flesh of cow, buffalo, sheep, and goats, in that order. In addition to this analysis technique indicated that8 (44.4) % of examined cattle meat samples were mixed with buffalo meat and10 (55.6) % of examined sheep meat samples were mixed with goat meat. As well as, most of examined (normal and mixed) meat samples were contaminated with Staphylococcus aureus bacteria. Discussion: It has been demonstrated that the highly conserved Mitochondrial Cytochrome cyt b gene primers, when used in the multiplex PCR methodology, provide a sensitive, straightforward, adaptable, and dependable method for the investigation of meat samples and meat products. Conclusions: The current study's conclusions state that the “mitochondrial cytochrome cyt b gene” is a useful marker for differentiating between normal and adulterated meat. It also states that Staphylococcus aureus was found in 16/52 (30.76%) of the normal meat samples and 12/18 (66.7%) of the adulterated meat samples, with a significant importance of P=0.046.

Phenotypic and Genotypic Profiles of Extended-Spectrum Beta-Lactamase-Producing Multidrug-Resistant Klebsiella pneumoniae in Northeastern Thailand.

The global emergence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae presents a significant public health threat and complicates antibiotic treatment for infections. This study aimed to determine the prevalence of ESBL-producing K. pneumoniae in a clinical setting, analyze their antimicrobial susceptibility profiles, and characterize both phenotypic and genetic determinants. A total of 507 non-duplicate clinical isolates of Enterobacterales were collected between 2019 and 2020, and third-generation cephalosporin resistance was screened by disk diffusion. Identification of K. pneumoniae was confirmed using biochemical tests and PCR with species-specific primers. Antimicrobial susceptibility testing was conducted using disk diffusion, and phenotypic ESBL production was confirmed using the combined disk method. Multiplex PCR detected ESBL genes (blaTEM, blaSHV, and blaCTX-M) and identified blaCTX-M groups. The genetic relatedness of ESBL-producing strains was assessed using the ERIC-PCR approach. Fitty-four isolates were confirmed as ESBL producers, all classified as multidrug-resistant (MDR). All ESBL-producing K. pneumoniae isolates exhibited resistance to ampicillin and cefotaxime, with high resistance rates for ciprofloxacin (98.2%), azithromycin (94.4%), piperacillin-tazobactam (88.9%), and trimethoprim (83.3%). Genotypic analysis revealed blaCTX-M was present in 94.4% of isolates, blaSHV in 87%, and blaTEM in 55.5%. The blaCTX-M-1 group was the most prevalent, accounting for 96.1% of isolates. Co-harboring of blaCTX-M, blaSHV, and blaTEM occurred in 42.6% of isolates, with co-carrying of blaCTX-M, and blaSHV was observed in 23/54 isolates. The ERIC-PCR analysis revealed 15 distinct types, indicating high genetic diversity. These findings highlight the urgent need for ongoing monitoring to control the spread of ESBL among K. pneumoniae and emphasize the importance of early detection and appropriate antibiotic selection for effectively treating infection caused by these pathogens.

Identification and molecular detection of the pathogen of Phalaenopsis leaf yellowing through genome analysis.

Moth orchids (Phalaenopsis spp.) are globally popular ornamental flowers. However, effective management strategies for Phalaenopsis leaf yellowing remain elusive, making the disease a challenging obstacle affecting moth orchids at various growth stages. This disease manifests as collar rot, leaf yellowing, leaf abscission, and eventually, plant death. The lack of effective management strategies is likely attributed to a limited understanding of the disease pathogenesis and pathogen dissemination pathways. Fusarium phalaenopsidis sp. nov. was established in this study to stabilize the classification status of Phalaenopsis leaf yellowing pathogens using molecular and morphological features. The genome of the holotype strain was sequenced and assembled, revealing its genome structures. Analyses of virulence-related elements, including transposon elements, secondary metabolite biosynthetic gene clusters, effectors, and secreted carbohydrate-active enzymes, shed light on the potential roles of three fast core chromosomes in virulence. Two species-specific primers were designed based on unique gene sequences of two virulence-related proteins through comparative genomics and BLAST screening. The specificity of these primers was validated using isolates of F. phalaenopsidis, non-target species in the Fusarium solani species complex, other Fusarium species complexes, and saprophytic fungi. These results are intended to accelerate the identification of the pathogens, facilitate the study of disease pathogenesis, and pave the way for elucidating pathogen dissemination pathways. Ultimately, they aim to contribute to the formulation of effective control strategies against Phalaenopsis leaf yellowing.

Resting Cysts of the Toxic Dinoflagellate Gymnodinium catenatum (Dinophyceae) Ubiquitously Distribute along the Entire Coast of China, with Higher Abundance in Bloom-Prone Areas

Blooms of Gymnodinium catenatum have occurred occasionally in different areas of China and caused tremendous economic loss and even threatened human health. Not only is G. catenatum an important harmful-algal-bloom (HAB)-causing species, but also the only gymnodinioid dinoflagellate known to produce paralytic shellfish poisoning toxins (PSTs). Due to the germination of resting cysts, which often initiates blooms, the distribution and abundance of cysts in sediments and particularly the confirmation of cyst beds are important information for understanding and predicting dinoflagellate blooms. In this research, 199 sediment samples were collected from China’s coastal seas, ranging from the Beidaihe in the Bohai Sea (BS) to the southernmost sample from the Nansha Islands of the South China Sea (SCS). TaqMan quantitative PCR (qPCR) assays with species-specific primers and probes were developed to specifically detect the distribution and abundance of cysts in the 199 samples. The detection revealed that G. catenatum cysts were widely present in the sediments (126 of the 199 samples), with 93.55%, 74.65%, 42.37%, and 50% of the samples detected positively from the BS, YS, ECS and SCS, respectively, and covering the vast sea area from Nansha Islands to the Beidaihe area. The single-cyst morpho-molecular identification in the samples from Beidaihe confirmed the existence of G. catenatum cysts in the BS, and the positive detections of G. catenatum cysts using the qPCR methods. While G. catenatum cysts were widely distributed in all four seas of China, the average abundance was relatively low (1.0 cyst per gram of wet sediment). Three samples from the East China Sea (ECS), however, contained G. catenatum cysts at a relatively higher level (23 cysts g−1 wet sediment) than other sea areas, suggesting a pertinence of cyst abundance to the frequent occurrences of G. catenatum blooms in the area during recent years. Collectively, for G. catenatum being such an important toxic and HAB-causing species globally, the ubiquitous distribution of its cysts along the coastal waters of China and higher abundance in the bloom-prone areas warns us of a risk that cyst beds, although currently low in abundance, may seed HABs in any and many sea areas of China at any forthcoming year, and particularly those areas with records of frequent HABs outbreaks in the past.

First Molecular Detection and Genetic Characterization of Tetratrichomonas buttreyi and Pentatrichomonas hominis in Donkeys in Shanxi Province, China.

Two species of trichomonads, Tetratrichomonas buttreyi and Pentatrichomonas hominis, are common intestinal parasites that can impact animal health and productivity. Severe infection by these parasites can lead to diarrhea and wasting in affected animals. Notably, P. hominis is known to cause diarrhea and has the potential to be transmitted between animals and humans. Donkeys hold significant economic importance in China's agricultural sector. However, whether donkeys are infected with T. buttreyi and P. hominis remains unknown globally. To address this gap in knowledge, 815 fecal samples were collected from donkeys in three representative regions in Shanxi Province, North China. Then, the presence and genetic characteristics of T. buttreyi and P. hominis were examined using species-specific PCR primers amplifying the small subunit ribosomal RNA genes. The overall prevalence was detected to be 25.4% (207/815) for T. buttreyi and 0.7% (6/815) for P. hominis in donkeys in Shanxi Province. All obtained P. hominis sequences were identified as genotype CC1. Genetic analysis revealed that all P. hominis isolates from donkeys were clustered into the same branch with isolates detected in humans, suggesting possible zoonotic transmission. This study is the first to report the occurrence and prevalence of T. buttreyi and P. hominis in donkeys globally. These findings expand the host range of trichomonads and improve our understanding of their genetic diversity and zoonotic potential, providing essential baseline data for the prevention and control of these parasites in donkeys in the region.

First report of oriental theileriosis in the Intermediate Zone, Sri Lanka: Is Theileria orientalis type 7 always apathogenic?

PurposeTheileriosis in cattle has a significant economic implication for dairy production globally. Thus, it is crucial to investigate the prevalence of bovine theileriosis, the causative agent and genotypes of Theileria species in dairy cattle in the Kurunegala District in the Intermediate zone, where the third largest population of dairy cattle in Sri Lanka is present and experienced a substantial reduction in the cattle population recently. MethodsSixty blood samples and background data were collected from three dairy farms in Galpokuna (n = 20), Koulwewa (n = 20), and Andigama (n = 20) areas. Haematocrit was used to identify anaemia while Giemsa-stained blood smears demonstrated the presence of piroplasms. A Fisher's Exact Test (p < 0.05) compared the prevalence of infection among age groups and farms. PCRs using species-specific primers designed to amplify regions of MPSP and 18 s rRNA genes followed by sequencing of selected amplicons allowed phylogeny of the species detected. ResultsAll three farms had semi-intensive farming practices keeping animals in a ‘closed’ setup with limited movement. Theileriosis prevalence in dairy cattle was 55.0 % with no difference among the farms (Koulwewa: 65.0 %, Galpokuna: 50.0 %, Andigama: 50.0 %). One-third of the cattle (33.3 %) were anaemic based on haematocrit results but most showed mild anaemia. Anaemia was significantly higher in calves (45.0 %) than in adults (25.0 %; χ2 = 5.743; p = 0.03) tested positive for theileriosis. None of the animals showed any other signs of theileriosis. PCR revealed the presence of both T. orientalis (pathogenic or benign) and T. annulata (pathogenic). The sequencing data revealed that the T. orientalis genotype present in Kurunegala District is type 7. ConclusionThis is the first report on T. orientalis and T. annulata in dairy cattle in the Intermediate zone of Sri Lanka. The type 7 of T. orientalis was more common showing severe anaemia in calves while mild anaemia or no anaemia in adults and sub-adults. In immunologically intact animals, the reported genotype of the parasite can persist asymptomatically for life, occasionally causing a relapse, particularly under stressful conditions like pregnancy, lactation, and rapid changes in environmental conditions. However, the susceptibility of calves for pathogenic and apathogenic genotypes of T. orientalis and the carrier status of asymptomatic animals needs further investigation.

Morphological and molecular characterization of Macrophomina phaseolina (Tassi) Goid isolates causing charcoal rot of cowpea (Vigna unguiculata (L.) Walp) in Ghana

The study of the variations in Macrophomina phaseolina, the causal agent of charcoal rot, is critical in understanding the ecology of the isolates and the epidemiology of the diseases they cause. This study sought to identify and characterize M. phaseolina isolates using both molecular and morphological approaches and to determine the virulence of the isolates. Macrophomina phaseolina was isolated from cowpea-diseased stem tissues. Eleven M. phaseolina isolates from the major cowpea growing areas (Manga, Nyankpala, Yendi, Damongo, Tumu, Wa, Akomadan and Ejura) were morphologically and molecularly characterized. DNA of the M. phaseolina isolates were amplified using species-specific PCR primers MpKF1 (5'-CCGCCAGAGGACTATCAAAC-3') and MpKR1 (5'-CGTCCGAAGCGAGGTGTATT-3') and sequencing of the M. phaseolina specific amplicons was obtained. This was followed by searching against the NCBI nucleotide sequence database. Isolates were tested for their pathogenicity on a susceptible cowpea cultivar (IT97K-499-35). Virulence of the M. phaseolina isolates was expressed as the percentage of sown seeds that expressed seed rot and damping-off symptoms 14 days after planting. All isolates were confirmed as M. phaseolina based on the amplicon size obtained, and the sequenced amplicons showed an average of 99% homology to the available M. phaseolina sequences. The radial growth of isolates and the percentage of seed rot and damping off they caused on the IT97K-499-35 cultivar varied significantly. The percentage seed rot and damping off were positively correlated (r = 0.74 and 0.75) with the radial growth and colour intensity of the isolates, respectively. Macrophomina phaseolina infection and disease expression may be predicted by the colour and colour intensity of isolates as well as cowpea genotypes. Isolate Mp_3Mc (dense black colour) was the most virulent while isolate Mp_6Ec (grey colour) was the least virulent, resulting in 76.7% and 26.7% seed rot and damping off, respectively. The three clades resolved through the phylogenetic tree matched the distinct colour types of M. phaseolina which correlated with virulence. Isolates of M. phaseolina are diverse and differ in their pathogenicity. Efforts must be geared towards developing cowpea varieties that have resistance to charcoal rot disease.